The Mechanism Of Direct Activation Of Angiotensin II Type 1 Receptor Induced By Mechanical Stress In Cardiomyocyte

The excessive activation of angiotensin II type 1 receptor (AT1-R) was critically involved in the initiation and development of cardiac hypertrophy evidenced by marked attenuation of pressure overload induced-cardiac hypertrophy attributing to the blockade or knockout of AT1-R. Recently published data revealed a new finding that AT1-R can be activated by mechanical stress independent of AngⅡand consequently induced cardiac hypertrophy, although the mechanism, upstream regulating factors and intracellular signaling transduction pathways remained unclear. Here we demonstrated that Ca2+/calmodulin-dependent protein kinaseⅡ(CaMKⅡ) plays a crucial role in the activation of AT1-R induced by mechanical stress independent of AngⅡin the development of cardiac hypertrophy.We firstly performed in vitro studies by using mechanical stretched-COS7 cells or HEK293 cells which both in lack of endogenous AngⅡ. Mechanical stress fail to induce hypertrophic responses in COS7 cells artificially expressed with AT1-R without CaMKII, indicating the critical role of CaMKII in the mechanical stress induced-activation of AT1-R.Next cardiac hypertrophy model was established in angiotensinogen gene knockout (ATG-/-) mice subjected to transverse aorta constriction (TAC) for 2 weeks. AT1-R and CaMKII were inhibited by treating the TAC ATG-/- mice with Losartan or KN93, respectively. At 2 weeks after TAC, the ATG-/- mice with KN93 treatment showed a less cardiac hypertrophy and unchanged hemodynamics, which were similar to that in the TAC mice treated with Losartan, than the TAC mice with vehicle treatment characterized by adaptive cardiac hypertrophy. Accumulated CaMKII and marked binding of CaMKII with AT-R were detected in mechanical stress induced-hypertrophied cardiomyocytes, which were significantly inhibited by Losartan. Mutation in the middle autoinhibitory sequence of CaMKII failed to induce the hypertrophy response elicited by mechanical stress, indicating a possible binding site with AT1-R in the middle autoinhibitory sequence.Calcineurin functioned as a key molecule in the intracellular signal transduction pathway. We therefore hypothesized that it may also play a critical role in the cardiac hypertrophy resulting from the directly activated AT1-R by extracellular mechanical stimuli. FK506, a specific inhibitor of calcineurin, significantly reversed the cardiac hypertrophy in ATG-/-mice imposed with TAC for 2 weeks, suggesting an essential role of calcineurin in the intracellular signal transducing the extracellular mechanical stimuli mediated by AT1-R.Part1CaMKⅡwas involved in the activation of AT1-R induce by mechanical stress independent of AngⅡObjective: To elucidate the role CaMKⅡplays in the activation of AT1-R induced by mechanical stress without the involvement of AngⅡ.Methods:COS7 cells transfected with the plasmids of wild type of CaMKⅡand AT1-R plasmids were cultured in silicon-based plates pre-coated with collagen and incubated in serum and antibiotic-free conditions for 24 hours before mechanical stretch to 120% for 8 minutes. Losartan (10-6mol/L) or KN-93 (10-9mol/L) pre-administered in the medium for 30 minutes. After mechanical stretch, the cells were collected for the extraction of total protein and RNA for the following experiments.Results:Mechanical stretch did not induce the upregulation of the phosphorylation of ERKs and reprogramming of ANP, BNP and SAA in COS7 cells without any transfection or only transfected with CaMKⅡalone because of lacking AT1-R. In COS7 cells enforced expressing AT1-R but without CaMKⅡ, mechanical stretch slightly upregulated the phosphorylation of ERKs and the reprogramming of ANP, BNP and SAA, which were largely upregulated in those doubly transfected by CaMKⅡand AT1-R.Conclusion:These evidences suggested that CaMKⅡmay play an important role in the activation of AT1-R induced by mechanical stress independent of AngⅡ.Part 2Mechanical Stress Elicited the Translocation of CaMKⅡfrom Cytoplasm to Membrane and Induced the Binding of CaMKⅡand AT1-RObjective:Although evidences showed that CaMKⅡis involved in the activation of AT1-R induced by mechanical stress directly, CaMKⅡis mainly located in cytoplasm which is disconnected with AT1-R locating in the membrane of cardiomyocytes. So we hypothesized that CaMKII may translocate to the membrane and interacted with the AT1-R in the condition of mechanical stress.Methods:ATG-/- mice imposed with TAC for 2 weeks were continuously administered with Losartan (3 mg/kg/day) or KN93 (3 mg/kg/day) by osmotic minipumps subcutaneously implanted in the back of ATG-/- mice. Transthoracic echocardiographic analysis and invasive hemodynamics measurement were performed at the end of 2ed week. Excised hearts were weighed, perfused with PBS followed by 4% polyformaldehyde for global morphometry and fixed in 10% formalin for histological analysis. Paraffin embedded hearts were sectioned at 4-μm thickness and stained with hematoxylin and eosin (H-E). Cardiomyocytes were chosen from each section at a high magnification, and cross sectional area (CSA) of cardiomyocytes was measured. Total RNA was isolated from the left ventricular (LV) tissues and reverse transcription and polymerase chain reaction was performed. Total proteins extracted from LV tissues were subjected to Western blot analysis for phosphorylation of ERKs. The amounts of CaMKII were examined after the membrane fraction was divided from the cytoplasm fraction. Extracted membrane proteins were used for Co-immunoprecipitation with an anti-AT1-R or anti-CaMKII antibody. Frozen slices of heart tissue of mice were incubated with anti-CaMKII or anti-AT1-R antibodies served as the primary antibody, and then with secondary antibodies. The fluorescence was observed under confocal microscopy.Results:Two weeks after TAC, although compensated cardiac hypertrophy characterized by increases in left ventricular (LV) wall thickness, global heart size, heart weight/body weight ratio (HW/BW) and cross-sectional area (CSA) of cardiomyocytes in LV section and decreases of LV cavity, was obviously formed and fractional shortening (FS) was preserved in all TAC mice, it was attenuated by KN93 as effectively as Losartan without impacting the BP and LVESP of TAC mice, consisting with the upregulation of phosphorylation of ERKs and reprogramming of ANP, BNP, SAA and SERCA2. Although data from Max dP/dT and contractibility index indicated that Losartan may be more effective on improving the contractibility of hearts of TAC mice, there were no statistical differences on the inhibitory effects of cardiac hypertrophy between KN-93 and Losartan, suggesting inhibition of CaMKII blocked the activation of AT1-R resultant in the cardiac hypertrophy. We employed the method of co-immuneprecipitation using both anti-CaMKII and anti-AT1-R antibodies. Western blot analyses of immunoprecipitate with anti-AT1-R antibody from membrane fraction of heart tissue of mice for CaMKII expression showed a combination of CaMKII with AT1-R, which was much more significant in the TAC mice than in sham-operated mice. Consisting with the results from co-immuneprecipitation, immunofluorescent staining with CaMKII and AT1-R revealed that much more CaMKII was located in cell-surface area and co-existed with AT1-R in cardiomyocytes of TAC mice than in sham-operated mice. Taken the data from co-immuneprecipitation and immunofluorescent staining into consideration, we found it is Losartan, but not KN93, that inhibited the combination of CaMKII and AT1-R.Conclusion:These findings suggested that mechanical stress elicited the translocation of CaMKII from cytoplasm to membrane and induced the binding of CaMKII and AT1-R. Although KN93 was more effectively inhibited the translocation of CAMKII from cytoplasm to membrane, it can hardly inhibit the combination of CaMKII and AT1-R as Losartan did.Part 3The binding sites of CaMKII with AT1-R in the mechanical stress stimulated-myocardiocytesObjective: To determine the binding sites of CaMKII with AT1-R in the mechanical stress stimulated-myocardiocytes.Methods:Three CaMKII mutants were constructed, which were depleted in N terminal (8N), in middle autoinhibitory sequences (δM), in C terminal (δC).We detected the phosphorylation of ERKs in the COS7 cells doubly transfected by AT1-R and each mutants of CaMKII before and after mechanical stretch for 8 minutes.Results:Significant differences were observed in the phosphorylation of ERKs in the COS7 cells doubly transfected by AT1-R and wild type,δC or 8N mutants before and after mechanical stretch, suggesting that CaMKII is essential in the activation of AT1-R and depletion of the C terminal and N terminal of CaMKII has no obvious effect on the function conducing activation of AT1-R. However, no significant differences in phosphorylation of ERKs were found in COS7 cells co-transfected with AT1-R andδM mutant of CaMKII before and after mechanical stretch which indicated that middle sequence of CaMKII is a critical site for the binding of CaMKII to AT1-R.Conclusion:The middle sequence of CaMKII is essential in mediating the activation of AT1-R and binding with AT1-R.Part 4Activation of AngiotensinⅡreceptor 1 by mechanical stress independent of AngiotensinⅡinduces cardiac hypertrophy through calcineurin pathwayObjective:Mechanical stress activates angiotensinⅡ(AngⅡ)type 1(AT1)receptor without the involvement of angiotensinⅡand therefore induces cardiac hypertrophy. However, the intracellular signal transduction pathway of AT1 receptor-mediated excellular mechanical signaling remains indeterminate.Methods:ATG-/- mice subjected to TAC for 2 weeks were continuously administered with Losartan (3 mg/kg/day) or FK506 (1mg/kg/day) by osmotic minipumps subcutaneously implanted in the back of the ATG-/- mice. Transthoracic echocardiographic analysis and invasive hemodynamics measurement were performed at the end of 2ed week. Excised hearts were weighed, perfused with PBS followed by 4% polyformaldehyde for global morphometry and fixed in 10% formalin for histological analysis. Paraffin embedded hearts were sectioned at 4-μm thickness and stained with hematoxylin and eosin (H-E). Cardiomyocytes were chosen from each section at a high magnification, and CSA of cardiomyocytes was measured. Total RNA was isolated from the LV tissues and realtime PCR was performed to detect the expression of ANP and SAA. Total proteins extracted from LV tissues were subjected to Western blot analysis for phosphorylation of ERKs.Results:We demonstrated that Losartan attenuated the cardiac hypertrophic responses and the upregulation of myocardial Ca2+ -dependent phosphatase calcineurin (CaN) in ATG-/- mice subjected to TAC for 2 weeks without an antihypertensive effect, indicating CaN is a downstream effector of AT1 receptor-mediated mechanical signaling inducing hypertrophic responses. Compared with Losartan, FK506, a specific inhibitor of CaN, similarly reversed the myocardial hypertrophy manifested with a preserved LVEF, less thicker ventricular wall thickness and enlargement of left ventricular cavity, decreased heart weight/body weight ratio and cross sectional area as well as the inhibition of up-regulated expression of fetal type genes including ANP and SAA, implicating an essential role of CaN in the mechanical stress induced-cardiac hypertrophy independent of AngⅡ.Conclusion:These findings collectively suggested that activation of AT1 receptor by mechanical stress independent of AngⅡinduces cardiac hypertrophy through CaN pathway.Conclusion1, CaMKII is involved in the activation of AT1-R induced by mechanical stress independent of AngⅡ.2, Mechanical stress elicited the translocation of CaMKII from cytoplasm to membrane and induced the binding of CaMKII and AT1-R, which enable AT1-R responding to the mechanical stress directly and consequently resulting in cardiac hypertrophy.3, The middle sequence of CaMKII is essential in mediating the activation of AT1-R and binding with AT1-R.4, Losartan, but not KN93, inhibited the combination of CaMKII and AT1-R induced by mechanical stress.5, Activation of AT1-R by mechanical stress independent of AngⅡinduces cardiac hypertrophy through calcineurin pathway.The potential application and novelty of this project1. We explored the mechanism of a new finding that mechanical stress activates AT1-R without the involvement of AngⅡ.2. The bindings and interaction between CaMKII and AT1-R is critically involved in the direct activation of AT-R induced by mechanical stress.3. Our findings indicated that mechanical stress promoted the translocation of CaMKII from cytoplasm to membrane, indicating a new function of CaMKII besides of its signaling transduction in cardiomyocytes.4. We found the anti-hypertrophied effect of Losartan partly attributed to its inhibition of the binding of CaMKII and AT1-R, which may provide new evidences for the development of ARBs in the cardioprotection.